Pth11 and MoSho1 are transported by MoEnd3-mediated endocytosis.

(A) Pth11:GFP was co-localized with FM4-64 in cytoplasm of germ tubes at 3 h. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (B) MoSho1:GFP was co-localized with FM4-64 in cytoplasm of germ tubes at 3 h. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (C) MoMsb2:GFP punctuate structures were not co-localized with FM4-64 marked endosomes in germ tubes. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (D) Appressorium formation assay with addition of LatB. Guy11 conidia significantly reduced appressorium formation after exposure to LatB for 30 min. The appressorium formation rates were recorded by observing 100 conidia for each sample and the experiment was repeated three times. (E) Pth11:GFP localization patterns with DMSO solvent and LatB treatment in germ tubes of Guy11 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5 μm. (F) MoSho1:GFP localization patterns with DMSO solvent and LatB treatment in germ tubes of Guy11 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5μm. (G) Pth11:GFP localization pattern in germ tubes of Guy11 and ΔMoend3 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5 μm. (H) MoSho1:GFP localization pattern in germ tubes of Guy11 and ΔMoend3 at 3 h. Insets highlight areas analyzed by line-scan. (I) Representative images of FRAP analysis for diffusion at Pth11:GFP localized regions in germ tubes of Guy11 and ΔMoend3. The fluorescence of Pth11:GFP significantly recovered at 35 s post-photobleaching in Guy11 but not in ΔMoend3. (J) Normalized FRAP curves of Pth11:GFP localized regions in Guy11 and ΔMoend3. 20 regions from different cells were subjected to FRAP analysis for each strain. Intervals: 5 s. (K) Representative images of FRAP analysis for diffusion at MoSho1:GFP localized regions in germ tubes of Guy11 and ΔMoend3. The fluorescence of MoSho1:GFP significantly recovered at 35 s post-photobleaching in Guy11 but not in ΔMoend3. (L) Normalized FRAP curves of MoSho1:GFP localized regions in Guy11 and ΔMoend3. 20 regions from different cells were subjected to FRAP analysis for each strain. Intervals: 5 s.